Systematic pharmacology-based strategy to explore the mechanism of Semen Strychni for treatment of papillary thyroid carcinoma

The aim of the study was to investigated the mechanism of Strychnos nux-vomica L. (Semen Strychni, SS) against papillary carcinoma thyroid (PTC) by combined of network pharmacology and experimental verification. By searching the TCMSP, SEA and SwissTarget Prediction database, the main active ingredients and related targets were obtained. Utilizing Venny 2.1.0 String database and Cytoscape 3.7.2 to screened the intersection target and constructed protein–protein interaction (PPI) network diagram. Using R 4.0.4 software carried out the enrichment analysis of GO and KEGG. HPLC was carried out using LC-20A modular HPLC system to identify the bioactive compound brucine present in SS. Molecular docking was performed using Discovery 2019 software. The inhibition rate was detected by CCK8 method. Western blot was used to detect the expression levels of brucine anti-PTC related pathway proteins. 14 active components were screened out, of which 4 main components showed tight relationship with PTC. SS may play the anti-PTC role by acting on two main pathways (TNF signaling pathway and MAPK signaling pathway) and mediating various biological functions. HPLC analysis revealed that brucine was a suitable marker for standardization of the SS. 4 active components exhibit strong binding energy with core protein. Brucine could significantly reduce the activity of BCPAP cells compared with isobrucine, stigmasterol, (+)-catechin. Brucine may reduce the protein expression levels of IL-6, VEGFA, JUN, TP53, 1L1B, PTGS2, BCL2, CASP3, CASP8, and CASP9 while increase the protein expression levels of BAD, cleaved-CASP3, cleaved-CASP8, and cleaved-CASP9 in BCPAP cells, respectively. The active components of SS against PTC mainly include isobrucine, stigmasterol, (+)-catechin, brucine. Among them, brucine exhibits the strongest anti-PTC activity in BCPAP cells, which may reduce the PTC-related protein expression levels. Therefore, SS may exhibits the anti-PTC activities through multiple targets and pathways.

Thyroid papillary carcinoma (PTC) is the most common type of differentiated thyroid carcinoma, accounting for 80-85% of the total incidence of thyroid malignant tumors 1,2 .In the past 30 years, the incidence rate of thyroid cancer in most regions of the world has continued to rise, and it has become the fastest growing solid tumor with incidence rate in the world 1,2 .According to the data of the Chinese National Cancer Registration Center (CNCHS), thyroid cancer in China will continue to grow at a rate of 4.5% every year.According to the final data which released by China Cancer Center (CCC) in 2017, the total incidence rate of thyroid cancer was about 10.16/105, ranking seventh in the incidence rate of malignant tumors 3,4 .Among them, PTC is the most common pathological type of thyroid cancer, accounting for more than 85% of thyroid cancer 5 .Most PTC patients have a good prognosis after traditional surgical treatment.Although the prognosis of most PTC patients is relative good, PTC will metastasize to the neck or distant lymph nodes in a few cases.The lymph nodes metastasized by PTC may invade important tissues such as blood vessels, nerves and trachea, resulting in increased difficulty in surgery, which may increase the risk of secondary or multiple operations for PTC patients, and finally affect the prognosis of patients 6,7 .
Strychnos nux-vomica L., (Semen Strychni, SS), as the mature seed of Yunnan Strychnos nux or Strychnos nux, is a commonly used Traditional Chinese Medicine (TCM) in clinical practice.It belongs to the liver and

Acquisition of intersection targets
Using R 4.0.4software 21 and Venny 2.1.0online analysis system (https:// bioin fogp.cnb.csic.es/ tools/ venny) to analysis the intersection target genes of active components related target genes and PTC related target genes, the common action target of disease and drug was obtained.Then using the Metascape database (http:// metas cape.org/), import the corresponding genes, and obtain Gene ID, the relevant genes of SS anti-PTC was finally acquired.

Construction of "component-target-disease" network diagram
The target genes corresponding to the active ingredients of SS and the PTC-related target genes were matched to obtain the common genes.That is, the key targets of SS against PTC was obtained.Nodes in the network represent components, targets and diseases respectively; the edge in the network is used to connect drugs and active components, active components and target genes.The whole network showed the relationship among drugs, active components and targets.The "component-target-disease" network diagram was constructed by using Cytoscape 3.7.2software 22 .

Construction of protein-protein interaction (PPI) network diagram
Input the intersection target genes obtained into the String online analysis platform (https:// cn.string-db.org/), select "Homes sapiens" as the species category, and obtain target interaction PPI network diagram.The results were exported in "TSV" format, and key target genes were screened by using Cytoscape 3.7.2software.

GO function enrichment analysis and KEGG pathway enrichment analysis
Using DAVID database (https:// david-d.ncifc rf.gov/ home.jsp) to carry out the GO function enrichment analysis and KEGG pathway enrichment analysis.After setting the threshold value to P < 0.05, take the top 20 GO term and KEGG channels, analyze them with clusterProfiler package of R 4.0.4software 23 , and draw bubble chart and column chart respectively.In addition, to validated the anti-PTC mechanism of SS across the key targets and multiple pathways, the KEGG mapper functional analysis was used to mark the target genes on the pathway associated with PTC.

Molecular docking verification
The SDF files with 2D structure of monomer compounds from SS was retrieved and downloaded respectively in PubChem database (https:// pubch em.ncbi.nlm.nih.gov/).Then the PDB file of core target protein structure was downloaded from RCSB database (https:// www.rcsb.org/).The Discovery Studio 2019 software was used to verify the docking result between the screened compound small molecules and the core target protein macromolecule with the highest degree.
HPLC analysis of SS and brucine SS were air-dried under laboratory conditions and then ground into small pieces.About 50 g of SS was soaked in ethanol solvent (200 mL) for 7 days and filtered with cotton plugs.The ethanol is evaporated under pressure, and the extract is concentrated using a rotary evaporator.Shimadzu LC-20A high performance liquid chromatography (HPLC) with dual solvent pump high pressure gradient system, SPD-20A photodiode array detector and automatic sampler are used for one-dimensional separation.Accurately weigh the total 95% ethanol extract of SS and brucine standard respectively, and dissolve them in methanol to make the final concentration 1 mg/mL.Using 0.5 μM organic membrane filtration and preparation for HPLC analysis.The gradient of binary mobile phase composed of ethanol solvent was used for chromatographic elution.The initial gradient condition is set as 5% ethanol, the flow rate is 1.0 mL/min, and the gradient increases to 100% ethanol within 60 min.The column temperature was maintained at 37 °C during the whole process.Shimadzu C18 column (250 mm × 4.6 mm, 5 μm), the detection wavelength is 254 nm.The volume of the total 95% ethanol extract of SS and brucine standard injection is 20 μL.
Cell culture BCPAP cell was cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin streptomycin at 37 °C and 5% CO 2 incubator.Cell growth was observed under inverted microscope, and cells in logarithmic growth phase were taken for subsequent experiments.

CCK8 assay kit
Grouping: different concentration of drug group: 2, 4, 8, 16, 32, 64, 128 µmol/L, 4 active compounds (isobrucine, stigmasterol, (+)-catechin, brucine) of SS respectively.Control group: the same volume of DMSO (0.01%).BCPAP cells were treated with 5 × 10 3 cells (100 μL suspension) was inoculated into 96 well plates.Place it in a 37 °C, 5% CO 2 incubator for 24 h, and then the cell monolayer will cover the bottom of the hole.The concentration of 4 active compounds added to the cells at 2, 4, 8, 16, 32, 64, 128 µmol/L in complete medium 100 μL, each with 3 double wells.Add 90 μL serum free DMEM and 10 μL CCK8 solution per hole, continue to culture for 2 h, and then measure the absorbance of each hole with the microplate reader at 450 nm wavelength.Repeat the experiment for three times.Cells were continued to cultured for 48 and 72 h, and measured the cell activity in each group at 24, 48, 72 h respectively.Cell viability was measured by CCK8 according to the manufacturer's instruction 24 .Measured the OD value of each well on the computer for the sample to be tested.The cell survival rate is expressed as a percentage, and calculate the half inhibition rate (IC 50 ).

Western blot assay kit
Protein blotting is a gold standard method used to identify and quantify specific proteins in complex mixtures extracted from cell or tissue lysates 25 .Western blot method was used to detect the expression levels of anti-PTC related proteins.Brucine with the final concentration of 16, 32, 64, 128 µmol/L was added to the culture dish of BCPAP cells, culture for 48 h, collect cells on ice, extract the total protein of each group of cells by RIPA

Statistical methods
The data obtained were expressed in x ± s, analyzed by SPSS 23.0 statistical software, compared between groups by one-way ANOVA, and the images were drawn by GraphPad Prism 9.0 software.Image J software was used for protein gray scale calculation, and P < 0.05 was considered as statistically significant difference.

Patient and public involvement
No patient involved.

Screening of main active ingredients
The flow chart of the study is shown in Fig. 1.In the present study, 62 chemical components were retrieved through TCMSP database, SEA database and SwissTarget Prediction database.Based on the complexity of data and sample size, DL ≥ 0.18 and OB ≥ 30% were selected as screening conditions.After removing the components that did not find action targets and duplicates, 14 active compounds were finally obtained.The analysis of active ingredient data shows that (+)-catechin, brucine nitrogen oxides have high OB values among these active ingredients, while brucine and strychnine, although their OB and DL values are low, are reported as high content and effective active ingredients in the comprehensive literature, so they are included together.Finally, 14 candidate active compounds of SS are determined (Table 1).

Screening of common targets
10,626 disease related targets were retrieved from Genecards database, and 86 components related targets of SS were screened from TCMSP database, SEA database and SwissTarget Prediction database respectively.Venn diagram shows that there are 74 common targets between diseases and drugs (Fig. 2A).Common targets were screened by R4.0.4 software and Cytoscape 3.7.2software respectively.According to the highest degree, the first 6 targets were IL6, VEGFA, JUN, TP53, 1L1B and PTGS2 (Table 2).

Construction of drug composition target disease network diagram
With TCMSP and Genecards database as platforms, the relationship between drug components and common targets, and the relationship between common targets and diseases are made into Excel tables, and input into Cytoscape 3.7.2 to generate relevant network diagrams.Since there are 4 components closely related to common targets between common targets and drugs, redundant drug components are deleted and a drug component target disease relationship network diagram is established.In this network diagram, red nodes represent chemical components, while blue nodes plays an anti-PTC role through multi components and multi targets (Fig. 2B).

Construction of target gene PPI network
Through the String online analysis platform, the interaction data of 74 targets are obtained, and the analysis is carried out using the Cytoscape 3.7.2software to obtain the PPI network (Fig. 2C).Using R 4.0.4software and Cytoscape 3.7.2software to calculate the frequency of target interaction respectively, which are IL6, VEGFA, JUN, TP53, 1L1B and PTGS2 in turn.These targets are the most critical 6 genes in the PPI network of SS anti-PTC related targets, which further explains that SS acts on these 6 targets, and the targets are interrelated, interacted and synergetic, playing a role in inhibiting tumor cell growth (Fig. 2D).

GO and KEGG enrichment analysis
Through the enrichment analysis of GO on the anti-PTC target of SS by R 4.0.4software, 105 biological processes with P < 0.05 were screened, and the first 20 important functional analysis data were visualized.It can be concluded that the biological processes involved in the anti-tumor metastasis targets of SS by GO biological process enrichment analysis mainly include response to xenobiotic stimulus, rhythmic process, vascular process in circulatory system, response to hypoxia, response to decreased oxygen levels, response to oxygen levels regulation of tube diameter blood vessel diameter maintenance, regulation of tube size, circadian rhythm.membrane raft, membrane microdomain, postsynaptic membrane, presynaptic membrane, integral component of presynaptic membrane, intrinsic component of presynaptic membrane, caveola, plasma membrane raft, integral component of postsynaptic membrane, intrinsic component of postsynaptic membrane.G protein-coupled amine receptor activity, nuclear receptor activity, ligand-activated transcription factor activity, neurotransmitter receptor activity, serine hydrolase activity, nuclear steroid receptor activity, G protein-coupled serotonin receptor activity, serotonin receptor activity, RNA polymerase II CTD heptapeptide repeat kinase activity, amine binding and so on (Fig. 3A).II promoter in response to stress, DNA-templated transcription in response to stress, embryo implantation, neuroinflammatory response, positive regulation of histone modification, fever generation, regulation of heat generation.transcription repressor complex, general transcription initiation factor binding, growth factor receptor binding, cytokine activity, cytokine receptor binding, ubiquitin protein ligase binding ubiquitin-like protein ligase binding, DNA-binding transcription repressor activity, RNA polymerase II-specific, DNA-binding transcription repressor activity TFIID-class transcription factor complex binding, MDM2/MDM4 family protein binding (Fig. 3B).It is suggested that the active components of SS may inhibit the formation of PTC by interfering with the above biological processes.Through enrichment analysis of KEGG signal pathway of SS target, 117 signal pathways with P < 0.05 were screened, and 20 important pathways were visualized by R 4.0.4software.The pathways involved in the anti-PTC target of SS mainly include lipid and atherosclerosis, IL-17 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, calcium signaling pathway, chemical carcinogenesis-receptor activation, kaposi sarcomaassociated herpesvirus infection, endocrine resistance, measles, TNF signaling pathway, breast cancer, prostate cancer, neuroactive ligand-receptor interaction, fluid shear stress and atherosclerosis, Th17 cell differentiation, pertussis, pancreatic cancer, serotonergic synapse, colorectal cancer, small cell lung cancer, cAMP signaling pathway (Fig. 3C).The main anti-PTC targets involve pathways mainly including kaposi sarcoma-associated herpesvirus infection, human cytomegalovirus infection, rheumatoid arthritis, IL-17 signaling pathway,   AGE-RAGE signaling pathway in diabetic complications, C-type lectin receptor signaling pathway, TNF signaling pathway, measles, fluid shear stress and atherosclerosis, lipid and atherosclerosis, inflammatory bowel disease, pertussis, leishmaniasis, MAPK signaling pathway, chagas disease, toll-like receptor signaling pathway, Th17 cell differentiation, yersinia infection, non-alcoholic fatty liver disease, Hepatitis B (Fig. 3D).These pathways play an anti-PTC role by regulating the process of tumor generation.In addition, annotated map of the key target genes locations of SS in anti-PTC related pathways was presented in Fig. 4. It was found that most of the key target genes are closely with TNF signaling pathway (Fig. 4A) and MAPK signaling pathway (Fig. 4B) in anti-PTC activities respectively.

Molecular docking results
The structure (SDF format) of each compound retrieved and downloaded from PubChem database was verified by molecular docking with the first 6 selected core targets, and the Discovery Studio 2019 software was used for analysis.The libdock scores was showed in Table 3.The libdock scores were higher, suggesting that it is more likely to be the key drug active molecule of SS in the treatment of PTC.The degree of targets (Fig. 5A) and the heatmap of 6 core targets (Fig. 5B) were presented.The active compounds are bound to the target mainly through

Experimental verification results
The results of HPLC HPLC analysis of methanol extracts of SS was carried out along with brucine standard (Fig. 9).The analysis revealed that brucine standard showed characteristic peak corresponding to SS.The retention time for SS was 15.930 (Fig. 9A) and for brucine standard was 15.801 (Fig. 9B) respectively.The results showed that brucine was a suitable marker for standardization of the SS.

The IC 50 values of active compounds in SS
The active compounds brucine, isobrucine, (+)-catechin and stigmasterol from SS with different concentrations showed certain inhibition rates on BCPAP cells after 24, 48 and 72 h of treatment, respectively.The IC 50 values of brucine at 24, 48 and 72 h were 0.313, 0.265, 0.201 µmol/mL respectively (Fig. 10A).The IC 50 values of isobrucine at 24, 48 and 72 h were 0.435, 0.375 and 0.309 µmol/mL respectively (Fig. 10B).The IC 50 values of (+)-catechin at 24, 48 and 72 h were 0.448, 0.377 and 0.351 µmol/mL respectively (Fig. 10C).The IC 50 values of stigmasterol at 24, 48 and 72 h were 0.366, 0.315, 0.302 µmol/mL respectively (Fig. 10D).Among them, brucine showed the strongest inhibition on the growth of BCPAP cells (IC 50 value was the lowest).Therefore, brucine was selected as the most effective compound of SS in subsequent experiments.It was revealed that brucine has a significant dose effect relationship on the inhibition of BCPAP cells but without time effect relationship, and the inhibition rate is the highest.www.nature.com/scientificreports/

Effect of brucine on the expression of apoptotic proteins
For further investigated the molecular mechanism of brucine on anti-PTC, the cancer related apoptotic proteins were measured.The study results showed that the expressions of BCL2, CASP3, CASP8, CASP9 proteins were significantly increased while BAD, cleaved-CASP3, cleaved-CASP8, and cleaved-CASP9 proteins were significantly decreased which administrated with varies concentration (16, 32, 64, 128 µmol/L) of brucine (Fig. 12A,B).

Discussion
In the present study, the active components of brucine, isobrucine, stigmasterol, and (+)-catechin from SS were predicted by network pharmacological method.It was reported that brucine exhibits a broad spectrum of anti-tumor effects.Brucine may inhibit cell migration, cell invasion and angiogenesis mimicry formation of human triple negative breast cancer cell line MDA-MB-231 26 .Previous study showed that brucine regulates  www.nature.com/scientificreports/Wnt/β-catechin signal pathway that can inhibit the growth and migration of colorectal cancer cell LoVo 27 , effectively inhibit the adhesion of liver cancer cells, and prevent the movement and invasion of HepG2 cells 28 .It was also found that brucine may significantly inhibit human leukemia K562 cell line and HL-60 cells, and induce apoptosis 29 .In addition, isobrucine, stigmasterol, (+)-catechin the three potential pharmacological components may also play a synergistic anti-tumor effect, which deserves further experimental study.It was reported that isobrucine exhibits most potent cytotoxicity to tumor cell lines of MCF-7 30 .It was also revealed that isobrucine may make a contribution to the anti-proliferation on HepG2 cells, although they are present in SS with small quantities 31 .Previous research found that isobrucine showed the potential anti-cancer activities of tumor cell lines of K562, HeLa and HEP-2 32 .Recent studies on stigmasterol rich plant extracts have shown that they have significant anticancer effects on various tumor cell lines by inhibiting cell cycle progression and inhibiting cell growth by regulating cell proliferation.Especially in skin cancer 33 , gastric cancer 34 , lung cancer 35 , and liver cancer 36 , stigmasterol plays a prominent role through different mechanisms, and its activity seems to be dose dependent.Catechin is natural phenolic compound which is a phytopharmaceutical with promising anticancer effects but poor bioavailability 37 .Catechin have shown effectiveness as anti-inflammatory and anti-cancer, mainly through its activity to alter the pathway by NF-ΚB, Nrf-2, and MAPKs 38 .Compared with chemotherapy drugs, these catechin nanoparticles have relatively low systemic toxicity and are promising drugs with low side effects for cancer treatment.The anticancer activity of catechins has been demonstrated in various in vitro and in vivo cancer models with different potential molecules 39,40 .Taken together, above 4 active compounds of SS showed relatively strong broad-spectrum anti-cancer activity.Through network pharmacological research, 6 key targets including IL6, VEGFA, JUN, TP53, 1L1B and PTGS2 were screened as important anti-PTC targets of SS.Through the construction of SS "drug-componentdisease" target network and target gene PPI network, it was revealed that the target sites coordinated the process of proliferation, apoptosis, differentiation and metabolism to inhibit tumor metastasis through interaction.It has been demonstrated that the IL6 blockade potentiates the anti-tumor effects of γ-secretase inhibitors in Notch3-expressing can be abrogated by the IL6R blocking antibody tocilizumab in breast cancer 41 .It was also suggested that mucoepidermoid carcinoma patients might benefit from combination therapy with an inhibitor of IL-6R signaling and chemotherapeutic agent such as paclitaxel 42 .In previous study, it was revealed that IL-6 exhibits a significant role in thyroid cancer progression and targeting IL-6 signalling may be helpful in clinical management of thyroid carcinoma (TC) patients with more aggressive tumour characteristics 43 .It was showed that the expression of CD30L/CD30 is accompanied by the expression of IL-6/IL-6R signal.It may be of clinical significance to evaluate the expression of IL-6 protein in PTC and MTC, because of the expression level is related to tumor invasiveness 44 .
VEGFA protein has the biological activity of inhibiting the formation of new blood vessels.When tumor cells appear, its expression level will generally rise greatly.Some studies have shown that VEGFA is related to tumor infectivity and tumor susceptibility 45,46 .Previous research found that VEGFA gene and its protein product is widely expressed and increased in PTC and colloid goiter 45 .In addition, it was revealed that the molecular status of VEGFA may play a significant role in the progression of PTC 46 .JUN protein may be used as a specific diagnostic biomarker/therapeutic molecular target of PTC 47 .It was reported that the c-Jun was associated with the presence of extra-thyroid invasion and degree of tumor infiltration, while the T allele and acetylated c-Jun also correlated with tumor stage 48 .P53 gene is a kind of tumor suppressor gene.It is a negative regulator in cell growth cycle, and is related to cell cycle regulation, DNA repair, cell differentiation, apoptosis and other important biological functions.One of the most powerful obstacles to cancer is the normal function of p53.As a tumor suppressor gene, TP53 gene is a ubiquitous mutant gene, which may be involved in the formation and development of thyroid cancer 49 .Previous studies indicated that, the prevalence of homozygous Arg TP53 genotype in adult patients with radiation related PTC is significantly reduced compared with sporadic PTC cases and the general population, which suggests that other TP53 allele combinations may lead to the risk of papillary thyroid cancer in individuals with late childhood exposure 50 .IL1B is a key mediator for PTC to increase the release of immune regulatory factor prostaglandin E2 and tumor necrosis factor induced gene 6 protein and the expression level of various chemokine genes 51 .It was reported that IL1B polymorphism is risk factor for thyroid carcinoma in a Chinese Han population 52 .In previous study, it was found that the mRNA levels of PTGS2 encoding prostaglandin-endoperoxide synthase 2 increased in PTC, and an increased consumption of arachidonic acid was observed, which forms the oncogenic lipid in PTC 53 .It has been demonstrated that PTGS2 gene is detected in a large part of human thyroid cancer, which highlights the possibility of inhibiting tumor growth through COX-2 inhibition 54 .In the present study, we revealed that the expressions of IL6, VEGFA, JUN, TP53, 1L1B and PTGS2 were decreased by regulated by increase of the dose of brucine, isobrucine, stigmasterol, and (+)-catechin respectively.
Apoptosis refers to the programmed death of the body under the macro regulation of genes to maintain the stability of the internal environment of cells 55 .Previous research revealed that the pathogenesis of cancer is closely related to cell apoptosis 56 .Caspase-3 protein is one of the most important apoptotic executors among the caspase family and a major effector factor in the process of cell apoptosis.Its activation marks the irreversible stage of apoptosis 57 .Caspase-8 protein plays an important role in the development and progression of cancer, activating various structural and regulatory proteins involved in caspase-3 division 58 .These proteins are crucial for cell survival and maintenance, mediating and amplifying cascade reactions during apoptosis.It was reported that in the mitochondrial apoptosis pathway, when it receives a signal that is activated, it will cause cyto-c to enter the cytoplasm, bind with apaf-1 to form an apoptotic body, and induce caspase-9 activation.Finally, caspase-9 will transmit apoptosis information to the apoptotic executing protein caspase-3, triggering an apoptosis response 59 .Previous studies have shown that the bad protein is expressed in various cells and can participate in the entire process of cell apoptosis through cell signal transduction pathways and interactions with members of the caspase family 60 .In addition, as an anti-apoptotic factor, the physiological function of bcl-2 is to suppress cell apoptosis and prolong cell life.Related research shows that bcl-2 overexpression in cells can lead to changes in the nuclear redox balance, which plays a protective role by causing the accumulation of glutathione in the nucleus and reducing the activity of caspase 61 .In the present study, we further investigated the protein expression levels of BAD, BCL2, CASP3, CASP8, and CASP9, respectively.The results showed that brucine may significantly reduced the protein expression levels of BCL2, CASP3, CASP8, CASP9, and significantly increased the protein expression level of BAD.Furthermore, it was reported that cleaved caspase is the activated form of caspase which usually exists in cells undergoing apoptosis 62 .Therefore, the cleaved CASP3, cleaved CASP8, and cleaved CASP9 were detected respectively.The results revealed that brucine may increase the protein expression levels of BAD, cleaved-CASP3, cleaved-CASP8, and cleaved-CASP9 in BCPAP cells.
Further analysis on GO biological process enrichment and KEGG signal pathway of SS anti-tumor related targets showed that multiple signal pathways were related to anti-tumor, including IL-17 signaling pathway, endocrine resistance pathway, breast cancer, prostate cancer, etc.Through comparison, it was found that the most important pathways related to anti PTC mainly including TNF signaling pathway and MAPK signaling pathway.Previous researches showed that the representative endocrine resistance 63 , TNF signaling pathway 64 and MAPK signaling pathway 65 play an important role in the intracellular signal transduction pathway at all stages of tumor genesis and development, not only mediating cell activity through their own signal transduction process, but also jointly mediating complex biological activities in cells.The biological processes involved in the anti-PTC target of SS contain response to xenobiotic stimulus, mononuclear cell differentiation, positive regulation of transcription from RNA polymerase II promoter involved in cellular response to chemical stimulus, regulation of transcription from RNA polymerase II promoter in response to stress etc.Through the GO biological process enrichment analysis and KEGG signal pathway analysis of the anti-PTC related targets of SS, it has demonstrated that SS exerts its anti-tumor effect through multiple targets and multiple pathways.
Molecular docking is a method of drug design based on the characteristics of receptors and the interaction between receptors and drug molecules.In recent years, molecular docking has become an important technology in the field of computer-aided drug research 66,67 .In the present study, it has been demonstrated that the 4 active components of SS, isobrucine, stigmasterol, (+)-catechin and brucine, could bind to the 6 core protein targets in varying degrees.Experimental verification mainly concluding CCK8 test and Western blot test.CCK8 test results showed that brucine could significantly reduce the activity of BCPAP cells compared with isobrucine, stigmasterol, (+)-catechin.Western blot result showed that brucine could reduce the protein expression levels of IL-6, VEGFA, JUN, TP53, 1L1B, PTGS2, BCL2, CASP3, CASP8, CASP9 while increase the protein expression levels of BAD, cleaved-CASP3, cleaved-CASP8, and cleaved-CASP9 in BCPAP cells as the increase of dose, respectively.

Conclusion
In the present study, the molecular mechanism effect of SS on anti-PTC was studied by screening the active components of SS and predicting the anti-tumor target, constructing the "component-disease-target" network diagram, constructing the PPI network, and conducting GO, KEGG analysis and other network pharmacological methods on the target.It was successfully predicted the main active components of SS anti-PTC effect, anti-PTC target and its related signal pathways and biological processes.It reflects the complex mechanism of multicomponent and multi-target effects of TCM on diseases.To our knowledge, this is the first application of in silico molecular docking to identify a small molecular and compounds from SS in treatment of PTC-related cells.At the same time, the manuscript also studied the expression of active ingredient brucine in SS on the target protein through experimental verification methods, proving that the active ingredient may act on PTC through related pathways, which provides a research basis for SS anti-PTC.

Figure 1 .
Figure 1.Flow chart of the systematic pharmacology-based strategy study.

Figure 2 .
Figure 2. (A) Venn diagram of the common target gene screening of SS and PTC-related targets.(B) Active ingredients of SS anti-PTC target network.(C) PPI network of SS on anti-PTC.(D) Key PPI network of SS on anti-PTC.

Figure 3 .
Figure 3. (A,B) SS prevention and treatment of PTC gene GO enrichment analysis bubble diagram.(C,D) KEGG pathway enrichment histogram chart of the active ingredients of SS treatment of PTC.

Figure 4 .
Figure 4. Annotated map of the target genes related the main active components of SS on PTC-related signaling pathways.(A) TNF signaling pathway.(B) MAPK signaling pathway.

Figure 5 .
Figure 5. (A) Bar diagram of corresponding core targets genes of 4 main ingredients based on the degree value.(B) Heatmap of main active ingredients of SS with 6 core target genes.

Figure 12 .
Figure 12.Effect of brucine on the expression of (A) apoptosis related proteins and (B) cleaved caspase proteins.*P < 0.05 was considered as statistically significant difference vs BCPAP cell.
lysis buffer assay kit.Measure the protein concentration by BCA method, gradually carry out sample loading, electrophoresis, membrane transfer, membrane washing and sealing.Add the corresponding primary antibody (1L1B, IL6, JUN, PTGS2, VEGFA, TP53, BAD, BCL2, CASP3, CASP8, CASP9, cleaved CASP3, cleaved CASP8, cleaved CASP9) in proportion of 1:1000 respectively, incubation at room temperature for 1 h according to instructions, overnight at 4 °C, add the secondary antibody (IgG-HRP-conjugated antibodies) after membrane washing, incubate in a shaking table for 2 h, and wash the membrane with TBST for 3 times, 10 min each time.ECL imaging system is used for luminous development.The experiment is repeated at least 3 times, and the gray value is measured and statistically analyzed.

Table 1 .
General information of active ingredients of SS.

Table 2 .
Corresponding core targets genes of 4 main ingredients based on the degree value.

Table 3 .
The results of molecular docking.